Process for producing collagenase

ABSTRACT

A NON-FLAGELLATED, NON-NOTILE STRAIN OF CLOXTRIDIUM HISTOLYTICUM (ATTC NO. 21000), WHEN FERMENTED UNDER CONVENTIONAL CONDITIONS FOR THE GROWTH OF CL. HISTOLYTICUM, YIELDS AN ELABORATION PRODUCT CHARACTERIZED BY COLLAGENASE AND PROTEOLYTIC ENZYME ACTIVITY AND INHIBITS THE GROWTH OF MICROBES SUCH AS STAPHYLOCOCCUS AUREUS AND ORGANISMS OF THE GENUS CLOSTRIDIUM. THIS COLLAGENASE MAY BE EMPLOYED IN THE FORM OF A TOPICAL OINTMENT FOR DEBRIDEMENT OF NECROTIC TISSUE OR AS AN INJECTABLE SOLUTION TO FACILITATE INTERNAL SLOUGHING AND READSORPTION OF PHYSIOLOGICALLY ANTAGONISTIC TISSUE.

United States Patent 6 3,705,083 PROCESS FOR PRODUCING COLLAGENASE Angelo J. Chiulli, Hempstead, and Edwin H. Wegman, Freeport, N.Y., assignors to Agricultural Biologicals Corporation, Lynbrook, N.Y. No Drawing. Filed July 8, 1966, Ser. No. 563,702 Int. Cl. C12d 13/10; A61k 19/00 US. Cl. 195-62 6 Claims ABSTRACT OF THE DISCLOSURE A non-fiagellated, non-motile strain of Clostrz'dium histolyticum (ATCC No. 21000), when fermented under conventional conditions for the growth of Cl. histolytz'cum, yields an elaboration product characterized by collagenase and proteolytic enzyme activity and inhibits the growth of microbes such as Staphylococcus aureus and organisms of the genus Clostridium. This collagenase may be employed in the form of a topical ointment for debridement of necrotic tissue or as an injectable solution to facilitate internal sloughing and readsorption of physiologically antagonistic tissue.

This invention relates to collagenase and to processes for producing the same. More particularly, this invention relates to a novel collagenase having superior properties for the debridement of necrotic tissue.

The healing process associated with infected wounds, dermal ulcers, second and third degree burns, and other conditions which produce dermal lesions in man and other mammals, is ordinarily quite slow. One of the characteristics of such lesions is the presence of dead tissue at the site of the lesion. It is necessary to remove this dead tissue in order to provide a healthy base for the growth of new tissue. Removal of this dead tissue can be accelerated by debridement, either surgical or enzymatic. Surgical debridement has the disadvantage that it is quite painful if all dead tissue is cut away, leaving exposed live tissue. Yet it is essential that all dead tissue be removed in order to facilitate the healing of the lesion and the growing of new tissue. Enzymatic debridement offers a less painful and more satisfactory procedure for the removal of dead tissue. Efficient enzymatic debridement requires the removal not only of the obviously necrotic tissue but of that material, usually on the periphery of the wound while apparently viable contains sub-clinical necrosis.

The mammalian tissue which prevents the sloughing of all necrosis and developing necrosis is connective tissue or collagen. This is a protein which is resistant to all mammalian enzymes so that sloughing is dependent on slow denaturation processes which change the collagen into a new form which can then be digested by local or systemic enzymes. In order then to remove undenatured collagen more rapidly, the patient must be provided with a collagenase, i.e., an enzyme which Will digest undenatured collagen.

The urgency of removal of necrosis is related to two important factors. First, this necrosis is prone to rapid microbial infection. In addition, this dead and dying tissue prevents the initiation of the healing processes which in man are granulation and epithelization.

The use of collagenase as a material for debridement has been proposed previously. However, the previously known collagenases have not been as efficient debridement agents as the novel collagenase of this invention. In some cases the prior collagenases possessed only collagenase activity without substantial activity against the other proteins present.

An object of this invention is to provide a novel collagenase which is more efficient as a debridement agent than the presently known collagenases.

Another object of this invention is to provide a nontoxic collagenase which causes substantially no side eifects.

A further object is to provide a novel process for producing the novel collagenase of this invention.

A still further object is to provide a topical ointment containing the novel collagenase of this invention for application to dermal lesions.

A still further object is to provide an injectable collagenase which is effective in the debridement of connective tissue in the internal parts of the body.

A still further object is to provide a novel collagenase which inhibits the growth of bacteria.

The novel collagenase of this invention is produced as an elaboration product of the fermentation of a newly discovered species of Clostridiumhistolyticum. which has no flagella and is therefore non-motile. This new strain of Cl. histolyticum has been isolated as a mutant in a culture of conventional flagellated Cl. histolyticum and has been deposited in the American Type Culture Collection as ATCC No. 21000.

Surprisingly, the collagenase elaborated by this new strain of microorganism has unexpected superior properties as a debriding agent. This new collagenase efliciently cleans away all necrotic tissue and a small surrounding area of mixed live and dead tissue, attacking both collagen and other proteins. This leaves a clean surface from which the growth of new healthy tissue can be initiated.

The new collagenase of this invention has an inhibitory action on the growth of microbes. This new collagenase in concentrations greater than about 0.1 mg./ml. inhibits the growth of various microbes such as Staph. aureus and organisms of the genus Clostridium. Dilution of the collagenase to concentrations of 0.1 mg./ml. or less permits growth of these bacteria.

The strain of Cl. histolyticum which has been found to give the new collagenase of this invention differs from the usual Cl. histolyticum characterized in Bergeys Manual of Determinative Bacteriology (7th edition, 1957, pages 690-691), in that this new strain has no flagella and is therefore non-motile.

Fermentation according to this invention is carried out under conditions which are conventional for the growth of Cl. histolyticum. Fermentation is carried out at about 32 to 37 C. (preferably 37 C.) for 21 to 26 hours in a nutrient medium which includes proteinaceous materials such as tryticase soy and proteose peptone, as well as vitamins and mineral salts such as magnesium sulfate, potassium phosphate monobasic, sodium phosphate dibasic, and ferrous sulfate. The proteinaceous material serves as a source of both nutrient carbon and nutrient nitrogen. The preferred pH of the fermentation medium 3 is between 6.5 and 7.5. Other fermentation media for the growth of Cl. histolyticum are known in the art and these may be substituted for the above described medium. The fermentation vessel is inoculated with a culture of Cl. histolyticum ATCC No. 21000, using conventional inoculation techniques. Prior to inoculation, the fermentation vessel and its contents are autoclaved at an elevated temperature and pressure, e.g. 121 C. and 15 p.s.i.g., for 30 minutes, and are then cooled to room temperature, in order to sterilize the medium.

The fermentation broth is centrifuged in order to separate the broth from the cells of the microorganism. The clear centrifuged Ibroth may be poured into a saturated aqueous solution of ammonium sulfate in order to precipitate the collagenase. This ammonium sulfate solution is held at a low temperature, not over 6 C. and usually about 4 C. This solution may contain about 500 grams per liter of salt, the exact amount varying depending primarily on the amount contained in the saturated solution at the particular operating temperature chosen. The collagenase material remains in this ammonium sulfate solution for a substantial length of time, say about 18 hours, at a temperature which is not allowed to rise above 6 C. The precipitate is filtered or centrifuged according to conventional techniques. The collagenase is cooled and placed in dialyzing tubing, generally made of cellophane, and dialyzed against running water for about 24 hours in order to remove salt. The presence of ammonia in the effluent water can be determined by Nesslers reagent. Dialysis is continued until the Nessler reagent test is negative.

The dialyzed enzyme is frozen and then freeze dried. This may be accomplished by transferring the enzyme from the dialysis tubing into trays in which the freezing and freeze drying take place. The freeze drier is maintained at a pressure of 250 microns or lower. While freeze drying temperatures may vary widely, the operation is speeded if a relatively high temperature, about 21 C., is maintained. The time in the freeze drier is generally about 18 hours. The freeze dried material is removed from the drying trays, and is then placed in polyethylene bags.

The freeze dried collagenase product must be sterilized. The inventors have found that this can be done efficiently with low collagenase losses by irradiation. Cobalt 60 is the preferred radiation source, although other radioactive isotopes can be used. Irradiation can also be accomplished by means of X-ray, although this is not a preferred procedure.

Irradiation according to this invention subjects the lyophilized collagenase to a total radiation dosage of at least 2.5 megarads and preferably about 4.5 to 5.5 megarads. The dosage is not allowed to exceed approximately 7 megarads as higher doses result in some decomposition of the enzymes. Irradiation may be carried out in conventional machinery for this purpose. The enzyme to be irradiated is contained in the aforementioned polyethylene bags, which are placed at such distance that the irradiation intensity is fairly uniform throughout the bag. Total irradiation time is about 120- 160 hours when the irradiation intensity is on the order of about .03 to .04 megarad per hour.

Collagenase produced according to this invention can be sterilized by other means, such as filtration, if desired.

The sterilized material is tested for both collagenase activity and proteolytic activity, using the ninhydrin test and the Azocoll test respectively. Standard test procedures as described in the art may be used for making these tests. For example, the Azocoll and ninhydrin test procedures may follow those described by Mandl et al., J. Clin. Invest. 32, 1323 supra. The material is also tested for sterility.

The sterility test is carried out by diluting the enzyme to 0.1 mg./ml., placing aliquots of the enzyme into two sets of tubes, one containing a nutrient medium and a small number (e.g. about to 10) of viable microbial cells and the other containing the nutrient medium but no cells. Standard test microorganisms such as Staph. aureus may be used for this purpose. If the collagenase is sterile, growth will take place in the tubes containing cells but not in the tubes to which no cells have been added. Dilution of the enzyme to about 0.1 mg./ml. or less is essential in making the sterility test, because the enzyme in greater concentrations may inhibit the growth of microorganisms.

The collagenase of this invention is conveniently applied as a topical ointment. For this purpose, collagenase may be incorporated in a conventional topical ointment medium such as petrolatum in concentrations ranging from about 0.1% to about 2% by weight of collagenase. A preferred ointment contains 0.5% of collagenase by weight of petrolatum. The ointments of this invention may also contain antibiotics if desired in order to combat infection at the site of the lesion. Collagenase ointment of this invention is applied directly to the lesion. The area is first preferably cleaned in order to remove any materials which may interfere with the action of the collagenase. This can be done with a sterile gauze pad saturated with sterile water or butfer having a pH of 7.0 to 7.5. The ointment may be put on a gauze dressing which is applied directly to the lesion. Application of the ointment should take place either every day or every other day. The gauze containing the oiintrnent is covered with a sterile dressing in preferred procedures.

The debridement activity of collagenase of this invention can be demonstrated by controlled experiments on laboratory animals such as guinea pigs. For test purposes burns of predetermined intensity are produced on the test animals. Application of a collagenase ointment of this invention to the burned surfaces results in remarkably efiicient debridement of burned tissue.

The ointment of this invention has been found to be stable over a period of 48 weeks at room temperature. However, the enzyme is heat labile, and temperatures appreciably in excess of room temperature are avoided. To assure long shelf-life, the ointment should be stored at a temperature not in excess of 24 C.

Collagenase may be used as an injectable to facilitate internal sloughing and reabsorption of physiologically antagonistic tissue. Injectable solutions may be used, for example, to speed the sloughing of operable prostate glands whose viability has been destroyed by injection of liquid nitrogen, to destroy the stroma of tumor masses, and to digest the collagen matrix of excess calcium deposits. Suitable injectable solutions may contain about 0.2 to 5% by weight of collagenase in physiological saline.

Collagenase produced according to this invention has a combination of proteolytic enzyme and collagenase activity which makes it uniquely effective in the debridement of tissue. This collagenase can be used alone for effective debridement of necrotic and moribund tissue, While prior collagenases when used alone give poorer results. The conjoint use of previously known collagenase and a proteolytic enzyme cannot be relied on for effective debridement, because the two enzymes are frequently incompatible.

Virtually no side effects are caused by the collagenase of this invention. Slight inflammation of surrounding tissue occurs occasionally; otherwise no side effects have been observed.

Collagenase of this invention inhibits the growth of microorganisms when present in concentrations of about 1 mg./ml. or greater. In many cases, particularly when bacterial infection has not set in, the use of antibiotics in conjunction with the novel collagenase is not necessary. However, the instant collagenase may be used with an antibiotic when desired. The inventors have also discovered that the growth inhibiting properties of the instant collagenase require its dilution to about 0.1 mg./ml. or less in making sterility tests as afore-described.

This invention will now be described in greater detail with respect to specific embodiments thereof, as illustrated in the examples which follow.

EXAMPLE 1 A strainof Clostridium. histolyticum ATCC No. 21000 is used to inoculate a seed medium containing 3% by weight of Trypticase soy broth, 1% by weight of proteose peptone, balance water, and having a pH between 6.5 to 7.5, which has been previously sterilized by auto claving in excess of 121.5 C. and 15 p.s.i. pressure for more than 15 minutes followed by cooling to room temperature. The volume of each seed flask is 250 ml. The seed flasks are incubated for 48 hours at 37 C. At the end of this period, samples of the fermentation medium are examined microscopically to examine for purity, and the proteolytic activity is measured by the Azocoll method to be hereinafter described.

Ten liters of fermentation medium is prepared having the following composition:

Eight liters of the above medium is charged to a 10 liter fermentation vessel.

The fermentation vessel is autoclaved in excess of 121.5 C. and 15 p.s.i. for 30 minutes and allowed to cool to room temperature. It is then inoculated with the seed flask cultures of Clostridium histolyticum, and incubated at 37 C. for 21 to 26 hours. The cells are separated from the broth by centrifuging the latter at 10,000 r.p.m. until clear broth is obtained. The broth is then poured into a precipitation drum containing a saturated solution of aqueous ammonium sulfate (approximately 500 grams per liter) at 4 C. and thoroughly stirred for ten minutes. The precipitation drum and contents are held at 4 C. for 18 hours. The precipitate is then filtered, collected, and placed in a cellophane dialysis tube and dialyzed against running water for 24 hours. During the final hour, water is allowed to stay in contact with the tube. The water is checked for ammonia with Nesslers reagent. If the reaction is positive, dialysis is continued for 3 more hours, and the water is again checked with 'Nesslers reagent. When the reaction is negative, the dialyzed precipitate is emptied into stainless steel lyophilizing trays, frozen, and put into a tray freeze drier. The freeze drier is maintained at a pressure no greater than 250 microns, and at a temperature of 21 C. Freeze drying time is approximately 18 hours. The freeze dried material is removed, milled gently, and placed in polyethylene bags. One bag is used for each tray. Each bag contains an average of 35 grams each of freeze dried collagenase.

The enzyme is sterilized by irradiation using a cobalt 60 source. A pair of bags, and additional small sample portions if desired, having a combined weight of about 75 grams, are placed in a container 4% inches in diameter and 5 inches high. The container is irradiated in an apparatus which includes a shielded enclosure, a cobalt 60 source, and a turntable rotating about a vertical axis passing through the radiation source. In a typical run, a container filled with enzyme is supported on the turntable with its base 7 inches above the floor of the enclosure and its axis 5 inches from the source. The average height of the container above the floor is the same as the height of the source. In a typical run, the container was irradiated for a total of 142 hours, receiving an average dose of 5.0 megarads, which varied from 4.77 megarads at the base of the container to 5.25 megarads at the middle.

Each sample of collagenase after irradiation is tested for collagenase activity, protease activity, and sterility.

Collagenase assay. Undenatured collagen is made for the collagenase as follows: Bovine Achilles tendon taken from a freshly slaughtered animal is stripped of fat and adventitia and then cut into pieces approximately A" square. These pieces are soaked successively in three solutions of M/ 15 aqueous dibasic sodium phosphate at 6 C. for 3 days in each solution. After the third soaking the tendon is soaked for 24 hours in tap water. The washed tendon is then soaked successively in three 25% aqueous potassium chloride solutions for three days in each solution. After the third potassium chloride soaking the tendon is soaked in tap water for one week and the water is changed daily. The supernatant solution is checked for the presence of chloride ion using the silver nitrate test. Water washing is continued until this test is negative. All of the above processing is done at a temperature no higher than 6 C. At the conclusion of washing, excess water is drained and the tendon chunks are lyophilized.

25 mg. of shredded undenatured collagen is placed in a test tube, and 0.1 m1. of a 0.1% aqueous solution of the lyophilized enzyme and 5 ml. of tris buffer are added. The tris buffer had the following composition:

Distilled water, q.s. to 200 ml.

A control tube A is prepared as above, except that the enzyme is omitted. A second control tube B is prepared as above, except for omission of the collagen (Tube B contains enzyme). A third control tube C is prepared as above, except that 0.1 ml. of 0.1% trypsin (Armour Pharmaceutical Co., Kankakee, Ill.) is added in place of the collagenase. Each of the four tubes is incubated at 37 C. for 24 hours. After incubation, 0.5 ml. of the clear supernatant is decanted from each tube and placed in a 10 ml. volumetric flask. To each volumetric flask are added 1 ml. of 1% aqueous ninhydrin solution and 1 ml. of 5% aqueous pyridine solution. The volumetric flasks are placed in a boiling water bath for 20 minutes. Sufficient distilled water is added to give a total volume of 10 ml. and the test solutions are read in a Klett photoelectric colorimeter at 570 millimicrons against the control. The collagenase activity is expressed in C units. One C unit represents the calculated optical density reading obtained when one mg. of enzyme acts on 25 mg. of collagen under the above conditions. The collagenase must have a minimum activity of 40,000 C. units per gram; otherwise it is discarded.

Protease assay.Azocoll, a hide powder preparation coupled to an azo dye, is made according to the procedure of Oakley et al., Journal of Pathology and Bacteriology, vol. LVIII, No. 2, pp. 227, 232 (1946). Proteolytic enzyme activity using Azcoll" reagent is determined according to the procedure of Mandl et al., Journal of Clinical Investigation, 32, 1323 (1953). The protease activity must be not less than 4000 Q units; otherwise the enzyme is discarded.

EXAMPLE 2 An ointment is prepared by mixing 5 grams of sterilized collagenase, prepared as described in Example 1, with 995 grams of white petrolatum.

7 EXAMPLE 3 An ointment is prepared by mixing the following ingredients:

Gm. Collagenase (Example 1) Strep-Combiotic (Pfizer) White petrolatum 985 Strep-Combiotic is a parenteral antibiotic in unit dosage form, containing 0.5 gram of streptomycin, 300,000 units of procaine pencillin G, and 100,000 units of buffered penicillin G, made by Charles Pfizer & Co., New York, NY. It is incorporated in the ointment to prevent non specific debridement.

EXAMPLE 4 guinea pigs ranging in Weight from 500 to 800 grams were anesthetized with sodium pentabarbital PB. Both sides of the animals were clipped with an electric clipper and then shaved. A circular area 5 centimeters in diameter was marked out on each side tangent to a line parellel with and /2 inch from the animals spine. A 125 ml. beaker of water was heated to 80 C. as measured with a mercury bulb thermometer immersed just below the top surface of the water. The marked area of the animal was held in contact with the surface of the water for 20 seconds. The burn area was wiped with denatured ethyl alcohol which was then allowed to evaporate.

A test ointment prepared as described in Example 3, and a control ointment consisting of petro'latum and Strep-Combiotic in the amounts stated in Example 3, but containing no collagenase, were evaluated.

Of the 20 animals used in this study, 16 animals were treated with collagenase ointment on one side and control ointment on the other. Of these, 8 received collagenase ointment on the right side. Two animals were treated with collagenase ointment on both sides, and 2 animals received control ointment on both sides.

The ointments were applied to each test animal daily. The ointments were spread on gauze pads which were placed in contact with the lesion. These were taped in place circumferentially and one additional length of tape was run around the animals neck to prevent dislodging. The ointments were coded so that the person administering them did not know whether he was administering a collagenase ointment or the control ointment. The order in which the animals were observed was varied from day to day. Prior to each observation, the lesion was gently rubbed with a cotton wad dipped in water. Treatment in this manner was conducted for 96 hours. At the end of this time, the animals were observed and the percentage of debridement was determined by measuring the debrided area. Percentages of debridement using both collagenase and control ointments, and the difierences between these two percentages of debridement for the 16 animals that received collagenase ointment on one side and control ointment on the other, are given in Table I.

TABLE I Percent debridement While this invention has been described in both the specification and the examples with reference to specific details and embodiments thereof, it is understood that these are by way of illustration rather than limitation, and the scope of the invention shall be measured only by the appended claims.

We claim:

1. A process for producing collagenase which comprises growing cells of the non-motile and non-fiagellated strain of Clostrz'dium histolyticum identified as ATCC No. 21000 under anaerobic conditions in a fermentation medium containing nutrient sources of carbon and nitrogen, harvesting the cells, and recovering collagenase from the nutrient medium.

2. A process according to claim 1 wherein said collagenase is recovered by precipitation with an inorganic salt followed by dialysis of the precipitate.

3. A process according to claim 1 wherein the recovered collagenase is irradiated with a radioactive source until sterile.

4. The method of claim 3 wherein said radioactive source is cobalt 60.

5. The process of claim 3 wherein the total amount of irradiation is in the range of 2.5 to 7 megarads.

6. collagenase produced by the process of claim 1.

References Cited UNITED STATES PATENTS 1,943,633 1/1934 Sperti 19568 3,201,325 8/1965 Barton 19566 3,267,006 8/1966 Hakim et al. 195-66 X OTHER REFERENCES Seifter et al., article in Methods in Enzymology, vol. V pp. 659-665 (1962).

Kazdobina et al., Chemical Abstracts 62, 70g (1964).

Desrosier et al., Radiation Technology in Food, Agriculture, Biology (1960) p. 354.

Harris, Pharmaceutical Microbiology (1964), p. 212.

LIONEL M. SHAPIRO, Primary Examiner US. Cl. X.R.

UNITE STATES PATENT orries ('IEZRTEWCATF, es? QDRREC'EWN Patent No. 3,705,083 Dated December 5, 1972-3 Inven Ansolo (hiulli et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col. 1, line L "Agricultural Biologicals" should be Advance Biofactures-- line 47 after "wound" insert --which---- (101. 2 line 58 "tryticase" should be --t ryptioase-- Signed and sealed this 13th day of November 1973.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. RENE D. TEGTMEYER Attesting Officer Acting Commissioner of Patents FORM PO-105O (10-69) USCOMMJDC 60376.4,"

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